Next-generation sequencing (NGS) is a driving force for many new and exciting applications, including cancer research, stem cell research, metagenomics, population genetics and medical research. While NGS technology is continuously improving, library preparation remains a process bottleneck for many labs and a limiting factor in the types of samples that can successfully generate NGS data.

As a result, capturing the maximum possible genomic information in an NGS library is both critical and challenging, when starting from limited or damaged DNA samples. Standard library preparation methods that apply to many sample types – such as circulating cell-free DNA, FFPE DNA, ancient DNA and ChIP-seq DNA – can result in quality-related problems. These problems limit the utility of the data generated, from low library yield or conversion rates to high percentages of reads derived from adapter dimer molecules.

Procedure

Library construction

QIAseq Ultralow Input Library Kits start with double-stranded DNA that has been fragmented enzymatically, chemically, mechanically or naturally. Kits use an optimized end-polishing reaction and a new Ultralow Input Ligation formulation, along with QIAGEN’s proprietary HiFi PCR Master Mix. This combination maximizes the conversion rate of sample DNA into the NGS library, while efficiently and evenly amplifying even high and low GC content regions of the genome. This protocol enables the highest possible yield sequencing library, free of adapter dimer contamination – starting from a little as 10−100 pg* of DNA input. Due to the kit’s flexible protocol, the same kit can also be used for higher DNA input amounts, including PCR-free library preparations from as little as 10 pg or as high as 100 ng of DNA input.
*Note that the genomic complexity necessary for a given experiment will vary depending on the genome size of the organism and the fraction of that genome included in the target region of interest.

Reaction cleanup

Following adapter ligation and library amplification steps, reaction cleanup and removal of residual adapter dimers can be achieved by using Agencourt AMPure XP beads – which enable easy automation on various high throughput automation platforms.

Optional amplification of library DNA

PCR-based library amplification is normally required if the input DNA amount is below 100 ng. This protocol is optional, and enables high-fidelity amplification of the DNA library using the QIAseq HiFi PCR Master Mix that is included in the kit.

Contents and accessories

Dual-barcoded, plate-format adapters are included with the 96-reaction size QIAseq Ultralow Input Library Kit. Each well in the 96-plex adapter plate contains a single-use adapter consisting of a unique combination of two eight-nucleotide identification barcodes. By combining one i5 barcode and one i7 barcode in each ready-to-use adapter, the 96 reaction QIAseq Ultralow Input Kits supports up to 96-plex pooling of libraries prior to sequencing.

While adapters are not included in the 12-reaction size kit, two sets of 12-plex adapters with a single six-nucleotide barcode can each be ordered separately (QIAGEN cat. nos. 180985, 180986).

Applications

QIAGEN QIAseq Ultralow Input Library Kits have been designed to be the definitive solution for generating high quality libraries from even very challenging NGS samples. Intended for NGS researchers who seek a single library prep kit compatible with a wide range of ultra-low, low and standard input fragmented DNA – QIAseq Ultralow Input Library Kits enable new insights by maximizing performance, particularly from limited and damaged DNA sample types. The streamlined, 2.5-hour protocol for generating libraries from fragmented DNA using QIAseq Ultralow Input Library Kits also enables straightforward automation on different liquid-handling platforms.